Fig 1.
Fluid shear stress induced erythroid differentiation in MEL cells.
(A) Representative images of Wright-Giemsa staining on MEL cells treated with shear stress (middle panel) and 2% DMSO (right panel) and control cells (left panel). Bars represent 10 μm. (B) The areas of the cells in different groups. (C-E) The mRNA expression levels of markers for erythroid differentiation, Hbb-b1 (C), GYPA (D), and GATA1 (E), as detected by quantitative RT-PCR. *: p<0.05, ***: p<0.001. n.s.: no significance.
Fig 2.
Fluid shear stress induced F-actin cytoskeleton remodeling in MEL cells.
(A-D) Representative flow cytometry images for MEL cells treated with 2% DMSO (A) and shear stress (C) for 24 h and stained with rhodamine phalloidin. The fold changes in F-actin content were shown in (B) and (D), respectively. As compared to control or static, *: p<0.05, **: p<0.01. (E, F) The confocal images for cells treated with 2% DMSO (E) and shear stress (F).
Fig 3.
Fluid shear stress upregulated E-Tmod41 expression in MEL cells.
(A-D) E-Tmod41 mRNA expression levels in MEL cells sheared for 3 h (A), 6 h (B), 12 h (C) and 24 h (D) as detected by quantitative RT-PCR. (E) The E-Tmod41 protein level in MEL cells sheared for 24 h as detected by Western blot (left panel) and its quantified data (right panel). GAPDH served as an internal control. As compared to static, *: p<0.05.
Fig 4.
Overexpression of E-Tmod41 increased F-actin content in MEL cells.
(A) E-Tmod41 expression in MEL cells infected with adenovirus, Ad-E-Tmod41, at 0, 25, 50, 75 and 100 MOI. β-tubulin served as an internal control. (B) Representative flow cytometry image (left panel) and the fold change of F-actin content (right panel) for MEL cells infected with Ad-E-Tmod41 and Ad-Null (50 MOI) and stained with rhodamine phalloidin. As compared to Ad-Null, *: p<0.05. (C) The F-actin images taken by confocal microscope (C).
Fig 5.
Knockdown of E-Tmod41 inhibited F-actin content in MEL cells.
(A, B) E-Tmod41 mRNA level (A, N = 2) and protein level (B) in MEL cells transfected with si-E-Tmod. (C, D) The flow cytometry data (C) and the confocal images (D) for MEL cells transfected with 100 nM si-E-Tmod and control siRNA and stained with rhodamine phalloidin. As compared to control, **: p<0.01.
Fig 6.
Fluid shear stress suppressed the expresson of miR-23b-3p and E-Tmod mRNA 3’UTR is the target of miR-23b-3p.
(A, B) The expressions of miR-23b-3p and its host gene, 2010111I01Rik, as detected by microRNA array analysis (A) and quantitative RT-PCR (B). (C) The putative binding site of miR-23b-3p (in red) is highly conservative in mammals. (D) Wild type E-Tmod 3’UTR with miR-23b-3p binding site and its mutant form (bottom). (E, F) The luciferase activities in MEL cells cotransfected with miR-23b-3p mimic and the luciferase reporter plasmids carrying either wild type (E) or mutant form of E-Tmod 3’UTR (F). As compared to control, *: p<0.05, n.s.: no significance.
Fig 7.
Overexpression of miR-23b-3p downregulated E-Tmod41 expression and contributed to F-actin cytoskeleton remodeling.
(A) The expression of miR-23b-3p in MEL cells transfected with miR-23b-3p mimic (100 nM) was verified by quantitative RT-PCR. (B, C) E-Tmod41 mRNA levels (B) and protein levels (C) in MEL cells transfected with miR-23b-3p mimic (50 and 100 nM). GAPDH served as an internal control. The quantified data was shown in (D). As compared to control, *: p<0.05. (E) The confocal images for MEL cells transfected with miR-23b-3p mimic and control mimic and stained with rhodamine phalloidin. The mean fluorescent intensities of the images were quantified and shown in (F).
Fig 8.
Knockdown of miR-23b-3p upregulated E-Tmod41 expression and contributed to F-actin cytoskeleton remodeling.
(A) The expression of miR-23b-3p in MEL cells transfected with miR-23b-3p inhibitor (100 nM) was verified by quantitative RT-PCR. (B, C) E-Tmod41 mRNA levels (B) and protein levels (C) in MEL cells transfected with miR-23b-3p inhibitor (50 and 100 nM). GAPDH served as an internal control. The quantified data was shown in (D, N = 2). (E) The confocal images for MEL cells transfected with miR-23b-3p inhibitor and control inhibitor and stained with rhodamine phalloidin. The mean fluorescent intensities of the images were quantified and shown in (F).
Fig 9.
Fluid shear stress regulated the activities of alternative promoters of E-Tmod.
(A) Schematic diagram of alternative promoters of E-Tmod. Boxes represent exons and lines represent introns. (B, C) The luciferase activities in MEL cells transfected with luciferase reporter plasmids driven by PE0 (B) or PE1 (C) promoters and subjected to shear stress for 12 h. As compared to static, *: p<0.05, **: p<0.01.
Fig 10.
Fluid shear stress induced erythroid differentiation and upregulated E-Tmod41 protein expression in embryonic erythrocytes.
(A) Representative Wright-Giemsa staining images for erythroblasts isolated from yolk sacs of day 10.5 embryos and subjected to shear stress for 24 h. The arrows in left panel indicate the erythroblasts. Bars represent 5 m. (B) The E-Tmod41 protein level was detected by Western blot (left panel) and the quantified result was shown in the right panel. As compared to static, **: p<0.01. (C) The protein level of glycophorin A were also detected by Western blot.